The hedgehog proteins are a family of extracellular signaling proteins that regulate various aspects of embryonic development both in vertebrates and in invertebrates (for reviews see Perrimon, N. (1995) Cell 80, 517-520 and Johnson, R. L., and Tabin, C. (1995) Cell 81, 313-316). The family includes members obtained from a variety of animals and includes Sonic, Desert, and Indian hedgehog. The most extensively characterized mammalian homolog is Sonic hedgehog (“Shh”), which is involved in diverse embryonic induction events, including the induction of floor plate and the establishment of ventral polarity within the central nervous system as well as proper anterior-posterior patterning of the developing limb (See Riddle, R. D., et al. (1993) Cell 75, 1401-1416; Echelard, Y. et al. (1993) Cell 75, 1417-1471; Roelink, H., et al. (1994) Cell 76, 761-775; and Roelink, H., et al. (1995) Cell 81, 445-455). In mediating these effects, Shh is believed to act both as a short range, contact-dependent inducer and as a long range, diffusible morphogen. Shh is expressed in the embryonic notochord, and induces floor plate formation at the ventral midline of the neural tube in a contact-dependent manner (See Riddle et al., and Roelink et al., Cell, 81:445 supra). Data suggest Shh can also act as a long range, diffusible morphogen, to promote subsequent differentiation of ventral neurons in a region-specific manner; e.g., dopaminergic neurons in the midbrain (Wang, M. Z. et al., (1995), Nature Med. 1, 1184-1188), and motor neurons in the spinal cord (Roelink et al, Cell 81: 445, supra).
Biochemical and genetic data suggest that the hedgehog receptor is the product of the tumor suppressor gene patched (Marigo, V., et al. (1996) Nature 384, 176179; Stone, D. M., et al. (1996) Nature 384, 129-134) and that other proteins, including smoothened (Alcedo, J., et al. (1996) Cell 86, 221-232), Cubitus interruptus or its mammalian counterpart gli (Dominguez, M., et al. (1996) Science 272, 1621-1625; Alexandre, C., et al. (1996) Genes & Dev. 10, 2003-2013), and fused (Therond, P. P., et al. (1996) Proc. Natl. Acad. Sci. USA 93, 4224-4228), are involved in the hedgehog signaling pathway. Induction of patched-1 and gli-1 expression are downstream markers of hedgehog signaling and serve as simple markers for hedgehog function. A second patched gene, patched-2 is also induced by hedgehog (Takabatake, T., et al., (1997) FEBS Letts. 410, 485-489). Hip, a transmembrane protein, has also been identified as a receptor for Hedgehog (PCT Publication W098/12326).
Shh is synthesized as a 45 kDa precursor protein that is cleaved autocatalytically to yield a 20 kDa N-terminal fragment that is responsible for all known hedgehog biological activity (“mature” Sonic hedgehog) and a 25 kDa C-terminal fragment that contains the autoprocessing machinery (Lee, J. J., et al. (1994) Science 266, 1528-1536; Bumcrot, D. A., et al. (1995) Mol. Cell Biol. 15, 2294-2303; Porter, J. A., et al. (1995) Nature 374, 363-366). The N-terminal fragment remains membrane-associated through the addition of a lipid tether at its C-terminus (Porter, J. A., et al. (1996) Science 274, 255-258; Porter, J. A., et al. (1995) Cell 86, 21-34). Recent biochemical data have identified this lipid tether as a cholesterol (Porter et al., Science 274, supra), the addition of which is catalyzed by the C-terminal domain during the autoprocessing step. Shh is also modified at its N-terminus with a palmitic acid (as described in PCT patent application Serial Number PCT US98/15923 and in Pepinsky et al., (1998) J. Biol. Chem., 273: 14037-14045). Lipid tethers restrict the tissue localization of the hedgehog signal and have presumably evolved as part of the mechanism for regulating short range-long range signaling by hedgehog.
Certain pathological conditions are caused by overexpression of hedgehog protein, including a variety of neural carcinomas. For these conditions, and others, it would be useful to develop versions of hedgehog that are capable of binding to hedgehog receptor proteins, but do not elicit signaling by hedgehog and, thus, serve as antagonists to hedgehog activity.